So you're going to a GATK workshop, and you've been selected to participate in a hands-on session? Fantastic! We're looking forward to walking you through some exercises that will help you master the tools. However -- in order to make the best of the time we have together, we'd like to ask you to come prepared. Specifically, please complete the following steps:

- Download and install all necessary software as described in this tutorial.

No need to install HTSlib, but all others are required. Note that if you are a Mac user, you may need to install Apple's XCode Tools, which are free but fairly large, so plan ahead because it can take a loooong time to download them if your connection is anything less than super-fast.

Speaking of long downloads, this one is also pretty big (740M), so again, don't leave it until last minute. This mini-bundle contains chromosome 20 of the human genome reference, a BAM file snippet and accompanying dbsnp + known indels files.

If you are attending the advanced hands-on session (if you're not sure, it's usually the one on the second or third day of the workshop), you'll need some extra files that aren't in the basic tutorial bundle. This add-on bundle is also quite large (870M) because it contains the complete human genome and a complete whole-genome callset. Note that this will take around 4G of space on your hard drive once it's uncompressed, so make sure you have plenty of space available on your machine.

At the start of the session, we'll give you handouts with a walkthrough of the session so you can follow along and take notes (highly recommended!).

Expected Result

This creates a GATKReport file called recal_data.grp containing several tables. These tables contain the covariation data that will be used in a later step to recalibrate the base qualities of your sequence data.

It is imperative that you provide the program with a set of known sites, otherwise it will refuse to run. The known sites are used to build the covariation model and estimate empirical base qualities. For details on what to do if there are no known sites available for your organism of study, please see the online GATK documentation.

2. Do a second pass to analyze covariation remaining after recalibration

Expected Result

This creates another GATKReport file, which we will use in the next step to generate plots. Note the use of the -BQSR flag, which tells the GATK engine to perform on-the-fly recalibration based on the first recalibration data table.

Expected Result

This generates a document called recalibration_plots.pdf containing plots that show how the reported base qualities match up to the empirical qualities calculated by the BaseRecalibrator. Comparing the before and after plots allows you to check the effect of the base recalibration process before you actually apply the recalibration to your sequence data. For details on how to interpret the base recalibration plots, please see the online GATK documentation.

4. Apply the recalibration to your sequence data

Action

Expected Result

This creates a file called recal_reads.bam containing all the original reads, but now with exquisitely accurate base substitution, insertion and deletion quality scores. By default, the original quality scores are discarded in order to keep the file size down. However, you have the option to retain them by adding the flag –emit_original_quals to the PrintReads command, in which case the original qualities will also be written in the file, tagged OQ.

Notice how this step uses a very simple tool, PrintReads, to apply the recalibration. What’s happening here is that we are loading in the original sequence data, having the GATK engine recalibrate the base qualities on-the-fly thanks to the -BQSR flag (as explained earlier), and just using PrintReads to write out the resulting data to the new file.

Have I done this right? These are the results I received. According to the tutorial if I saw the line that had the word "Walker" in it, then I did it right. But I'm not sure if I'm right or wrong because CountReads gave me the number of reads counted (2075853)

http://www.broadinstitute.org/gatk/guide/topic?name=tutorials
in Step 3, the example of code still has the deprecated walker
-T AnalyzeCovariants
which when used generates this,
"ERROR MESSAGE: Walker AnalyzeCovariates is no longer available in the GATK; it has been deprecated since version 2.0 (use BaseRecalibrator instead; see documentation for usage)"

I am a complete newb. Even with help and support from my lab mates, I need to read your materials. I was sent by the GATK Guide Book (page 10; section 4) to Dropbox location https://www.dropbox.com/sh/e31kvbg5v63s51t/ajQmlTL6YH where I picked up ReduceReads.pdf
On page 11 of that document there are ten graphs. The resolution of the .pdf file is so low that I cannot read the legends on the left side and bottom of these ten graphs. Could you point me to the high resolution version of this .pdf ?